Introduction
========
Methods and reagents is a unique monthly column that highlights current
discussions in the newsgroup bionet.molbio.methds-reagnts, available on the
internet. A commonly occurring theme on the net is the recovery of DNA, and
this month's column discusses the pros and cons of various methods used to
extract DNA fragments directly from agarose gels. For details on how to
partake in the newsgroup, see the accompanying box.

DNA electrophoresed through agarose gels is frequently used as a primary or
re-amplification template for the polymerase chain reaction (PCR) as well as
for hybridizations, sequencing, ligations, and many other molecular
techniques. To recover the DNA, the band of interest is excised with a sterile
scalpel and the DNA extracted by various means. Common practices include
organic extraction with phenol and chloroform, use of electroelution devices,
`freeze and squeeze' methods, `crush and soak' methods and a few different spin techniques.

The freeze and squeeze technique involves freezing the gel piece in liquid
nitrogen within a micropipet tip [1] or centrifuge tube [2], and spinning out
the liquid by centrifugation, while the crush and soak method involves adding
buffer to the agarose slice before squashing it with a glass rod. The slurry is
placed at 37 degrees C for several hours before the sample is centrifuged
through siliconized glass wool or non-toxic polyallomer fibers.

Electroelution
========
An electrophoresis unit [3] or a mini-gel system can be used for DNA extraction
by placing the gel fragments inside a dialysis bag along with electrophoresis
buffer so that, when electroeluted, the trapped DNA can be recovered by
precipitation. This can also be accomplished by using a centrifuge tube
mini-unit [4-7], or microtiter dish with capillary tubes that span two of the
titer-dish wells.

Another way is to cut a small trough ahead of the migrating DNA band and to
electrophoretically elute the DNA onto diethylaminoethyl (DEAE)-cellulose paper
[8], dialysis tubing [9], affinity membrane [10] or into a dead space in the
gel containing 0.3 M sodium acetate pH 6.0, 10 % sucrose [11]. The problem
with this method is that the gel must be visualized with UV and constantly
monitored to ensure collection of the sample.

Resin binding
========
A very popular method is to bind the DNA to silica particles by using
commercially available binding resins, diatomaceous earth or glass fibers.
This method was previously described in Methods and reagents [see TIBS
19,182-183]. Briefly, fragments from the gel are placed in a tube together
with a chaotropic substance and a binding matrix, usually composed of a
silica-based resin. The DNA is quickly bound to the resin and the complex is
washed several times with a 70% ethanol. The DNA-matrix is centrifuged out of
suspension, dried and the DNA is eluted in a small volume of sterile water.

Using this technique, DNA fragments up to 15 kb in length can be subcloned.
However, the efficiency of recovery drops dramatically when the size of the
fragment is larger than 6 kb. This presumably occurs because linear fragments
bind to more than one particle at once, causing them to be physically sheared
when pipeted.

Spin techniques
========
Another useful method is purification by placing the gel slice within a
microfuge tube containing a membrane with a small pore size, such as a Costar
Spin-X microfuge tube fitted with a 0.22 um filter [12]. Alternatively, gel
slices composed of low melting point (LMP) agarose can be placed in a microfuge
at 70 degrees C until the gel has melted. The molten agarose is then quickly
frozen and thawed, and the tube centrifuged for 5 min. This technique has the
advantage that very large fragments of DNA can be recovered with minimal
damage, especially if care is taken to expose it to long-wave UV radiation (366
nm) for only short periods of time [13].

Netters have reported problems with this method - the precipitated material
forms a pellet that does not easily dissolve in water, making it difficult to
work with. This can be rectified by not precipitating, but using the
supernatant directly instead.

Spin techniques may also cause co-elution of contaminants that could inhibit
DNA ligase and presumably other enzymes, or perhaps interfere with
transformation. In addition, the yield of DNA is usually not more than 60-70
%. Some netters are enthusiastic about this technique, however, claiming that
it works very consistently if the procedure is optimized [14].

GELase
========
Contaminating LMP agarose can also be removed by heating the gel slice to 65
degrees C for 10 min, lowering the temperature to 40 degrees C, and then adding
GELase[TM] (available from Epicentre Technologies), a combination of enzymes
including beta-agarase, which degrades the agarose into multimeric subunits.
[15,16] This technique generally gives high yields of DNA, but can only be used
with the high-grade LMP agarose because it has fewer sulfate groups, which
inhibit GELase activity. Unfortunately, this method is time consuming since
the digestion takes several hours or overnight. In addition, the DNA sample
may still require phenol extraction and precipitation.

There may also be some further problems with the method, since one netter
reported that DNA gel-purified with beta-agarase cannot be amplified by PCR,
while another reported that DNA fragments purified in this way could not be
enzymatically labeled by nick translation for subsequent use as a probe in a
hybridization experiment. The problem was only corrected by switching to a
recovery protocol that didn't use agarase.

Syringe squeeze
========
All the above protocols are time consuming. A faster and easier way is the
syringe-squeeze method of Li and Ownby [17], which can give 90-100% recovery in
less than 30 sec. However, the down side is that DNA may not be sufficiently
purified from contaminating agarose or buffer components for further
manipulations. For example, one netter complained that DNA extracted in this
way could not be ligated efficiently without an extra step of phenol/chloroform
extraction.

Summing up
========
Netters have found that the best choice of extraction procedure is highly
dependent on what the DNA is to be used for afterward. Typically, methods
involving extraction with organic solvents, electroelution, or binding of the
DNA to silica particles or ion-exchange resins give quite pure DNA, but yields
are relatively low. The apparently poor yield of DNA recovered by the spin
techniques has been found to be clean enough for ligating directly from the
eluate without organic extraction or any other further manipulation. On the
other hand, high-yield techniques tend to be problematic in enzyme reactions.
Clearly, there is a distinct trade-off between recovery and the purity of the
DNA sample.

========
Any statements made by the author are not meant to advocate the use of a
particular commercial product or endorse any company. All opinions are
those of the author and do not reflect the opinion of the National Cancer
Institute or the National Institutes of Health.

Copyright: This manuscript is not copyrighted by Elsevier Publishing Company.
However, you may not reproduce any portion for resale or edit the text for
redistribution, sale, or otherwise without written permission from the author.

You can find this at the World Wide Web (WWW) Uniform Resource Locator (URL)
ftp://ftp.ncifcrf.gov/pub/methods/TIBS/sep94.txt

Any reference to this column must be cited as the following published article:
Hengen, P. N. 1994. Methods and reagents - Recovering DNA from agarose gels
Trends in Biochemical Sciences 19(9):388-389.